Colorimetric and fluorescent methods for sensing of oligonucleotides

Abstract

Methods and kits are provided for detecting the presence or absence of target nucleic acid sequences in a sample. The methods and kits involve the use of metal nanoparticles and the electrostatic interactions between the metal nanoparticles and nucleic acid molecules. The methods rely upon the differential interaction of ss-nucleic acids and ds-nucleic acids with the metal nanoparticles. A colorimetric detection approach utilizes the ability of ss-nucleic acids electrostatically associated with metal nanoparticles in a colloidal suspension to stabilize them against aggregation. A fluorescent approach involving tagged ss-oligonucleotide probes translates the differential adsorption of ss-nucleic acids on metal nanoparticles to differential quenching of a fluorescent tag on probes that have not hybridized with targets.

1 . A method for detecting presence or absence of a target nucleic acid in a test solution comprising: 
 combining at least one single-stranded oligonucleotide probe with a test solution potentially including a target nucleic acid to form a hybridization solution, wherein the at least one single-stranded oligonucleotide probe and the test solution are combined under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution;    exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow the at least one single-stranded oligonucleotide probe that remains unhybridized after said combining to associate electrostatically with the plurality of metal nanoparticles; and    determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with one or more of the plurality of metal nanoparticles, wherein hybridization to the target nucleic acid or electrostatic association with one or more metal nanoparticles is indicated by an optical property of the hybridization solution.    
     
     
         2 . The method according to  claim 1 , wherein exposing comprises: 
 contacting the hybridization solution with a suspension comprising the plurality of metal nanoparticles, and    adding a salt solution.    
     
     
         3 . The method according to  claim 2  wherein the plurality of metal nanoparticles are introduced into the hybridization solution at a concentration of between about 1 and about 100 nM.  
     
     
         4 . The method according to  claim 2  wherein the salt solution comprises a Na +  concentration of between about 0.01 and about 5 M.  
     
     
         5 . The method according to  claim 2  wherein said contacting and adding are carried out simultaneously.  
     
     
         6 . The method according to  claim 2  wherein said contacting and adding are carried out in succession.  
     
     
         7 . The method according to  claim 6  further comprising a delay between said contacting and adding steps.  
     
     
         8 . The method according to  claim 1  wherein at least one single-stranded oligonucleotide probe is present in the hybridization solution at a concentration of about 1 to about 10,000 nM.  
     
     
         9 . The method according to  claim 1  wherein said determining comprises: 
 detecting a color change of the hybridization solution after said exposing, whereby a color change indicates substantial aggregation of the plurality of metal nanoparticles in the presence of a target nucleic acid.    
     
     
         10 . The method according to  claim 7  wherein said detecting comprises comparing the color of the hybridization solution to one or more precalibrated control solutions.  
     
     
         11 . The method according to  claim 7  wherein said detecting is carried out by naked eye observation of a user.  
     
     
         12 . The method according to  claim 1  wherein the at least one single-stranded oligonucleotide probe is coupled to a fluorescent label.  
     
     
         13 . The method according to  claim 12  wherein the fluorescent label is selected from the group consisting of an organic dye, semiconductor quantum dots, lanthanide atom-containing complexes, and a fluorescent protein.  
     
     
         14 . The method according to  claim 12  wherein the optical property is a fluorescence spectra or the magnitude of a fluorescence peak.  
     
     
         15 . The method according to  claim 12  wherein said determining comprises: 
 detecting a substantial reduction in fluorescence caused by the fluorescent label after said exposing, which indicates quenching of fluorescence by probes electrostatically associated with one or more of the plurality of metal nanoparticles, thereby indicating the absence of a target nucleic acid.    
     
     
         16 . The method according to  claim 12  wherein the test solution comprises a plurality of different target nucleic acids and the at least one oligonucleotide probe comprises a corresponding plurality of different oligonucleotide probes, each of the plurality of different oligonucleotide probes having a distinct fluorescent label.  
     
     
         17 . The method according to  claim 1  wherein the plurality of metal nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles, and combinations thereof.  
     
     
         18 . The method according to  claim 1  wherein hybridization complexes in the hybridization solution do not significantly associate electrostatically with the plurality of metal nanoparticles.  
     
     
         19 . The method according to  claim 1  wherein the target nucleic acid is DNA.  
     
     
         20 . The method according to  claim 1  wherein the target nucleic acid is RNA.  
     
     
         21  The method according to  claim 1  wherein the target nucleic acid is obtained from a human, a virus, a bacteria, an animal, an insect, or a plant.  
     
     
         22 . The method according to  claim 1  wherein target nucleic acid is synthetic, natural, or structurally modified DNA or RNA.  
     
     
         23 . The method according to  claim 1  wherein the target nucleic acid comprises a nucleic acid molecule coupled to a protein or polypeptide.  
     
     
         24 . The method according to  claim 1  wherein the target nucleic acid is a product of a polymerase chain reaction.  
     
     
         25 . The method according to  claim 24  wherein the test solution is obtained from a polymerase chain reaction solution, said method further comprising: 
 denaturing the product of the polymerase chain reaction prior to said combining.    
     
     
         26 . The method according to  claim 1  wherein each of the at least one oligonucleotide probes is between about 10 and about 50 nucleotides in length.  
     
     
         27 . The method according to  claim 1  wherein at least one oligonucleotide probe comprises structurally modified DNA or RNA.  
     
     
         28 . The method according to  claim 1  wherein the at least one oligonucleotide probe comprises a nucleotide sequence that is 100 percent complementary to the target nucleic acid.  
     
     
         29 . A method of quantifying the amount of a target nucleic acid is a test solution, said method comprising: 
 providing first and second control solutions each containing known but different amounts of a target nucleic acid;    performing the method according to  claim 1  on the first and second control solutions and the test solution, said performing comprising measuring the optical property of the hybridization solutions formed using the test solution and the first and second control solutions; and    calculating the quantity of the target nucleic acid in the test solution relative to the quantity of the target nucleic acid present in the first and second control solutions.    
     
     
         30 . The method according to  claim 29  wherein the optical property is the color of the hybridization solution.  
     
     
         31 . The method according to  claim 29  wherein the at least one oligonucleotide probe each comprises a fluorescent label and the optical property is a fluorescence spectra or the magnitude of a fluorescence peak caused by the fluorescent label.  
     
     
         32 . A method of quantifying the amount of a target nucleic acid is a test solution, said method comprising: 
 performing the method according to  claim 1  on the test solution, said performing comprising measuring the optical property of the hybridization solutions formed using the test solution; and    determining the quantity of the target nucleic acid in the test solution using the measured optical property and a calibration curve of measured optical property versus quantity of target nucleic acid.    
     
     
         33 . A method for detecting a single nucleotide polymorphism in a target nucleic acid molecule, said method comprising: 
 combining (i) a test solution comprising a target nucleic acid molecule and (ii) at least one first single-stranded oligonucleotide probe comprising a nucleotide sequence that hybridizes to a region of the target nucleic acid molecule that may contain a single-nucleotide polymorphism, to form a test hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one first single-stranded oligonucleotide probe to form at least one hybridization complex;    combining (i) a control solution comprising the target nucleic acid molecule and (ii) at least one second single-stranded oligonucleotide probe comprising a nucleotide sequence that hybridizes perfectly to a region of the target nucleic acid molecule that does not contain a single-nucleotide polymorphism, to form a control hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one second single-stranded oligonucleotide probe to form at least one hybridization complex;    exposing the test and control hybridization solutions, while maintaining the hybridization solutions at a temperature that is between the melting temperature of the at least one first single-stranded oligonucleotide probe and the melting temperature of the at least one second single-stranded oligonucleotide probe, to a plurality of metal nanoparticles under conditions effective to allow unhybridized probes in the hybridization solutions to electrostatically associate with the metal nanoparticles; and    determining whether an optical property of the test and control hybridization solutions are substantially different, indicating the presence of the single nucleotide polymorphism in the target nucleic acid molecule.    
     
     
         34 . The method according to  claim 33  wherein exposing comprises: 
 contacting each of the test and control hybridization solutions with a plurality of metal nanoparticles, and    adding a salt solution to each of the test and control hybridization solutions.    
     
     
         35 . The method according to  claim 34  wherein the plurality of metal nanoparticles are introduced into each of the test and control hybridization solutions at a concentration of between about 1 and about 100 nM.  
     
     
         36 . The method according to  claim 34  wherein the salt solution comprises a Na +  concentration of between about 0.01 and about 5 M.  
     
     
         37 . The method according to  claim 34  wherein said contacting and adding are carried out simultaneously.  
     
     
         38 . The method according to  claim 34  wherein said contacting and adding are carried out in succession.  
     
     
         39 . The method according to  claim 38  further comprising a delay between said contacting and adding steps.  
     
     
         40 . The method according to  claim 33  wherein at least one first and second single-stranded oligonucleotide probes are present in the test and control hybridization solutions at a concentration of about 1 to about 100,000 nM/ml.  
     
     
         41 . The method according to  claim 33  wherein the optical property of the test and control hybridization solutions is their color, whereby a difference in color indicates presence of the single nucleotide polymorphism in the test solution.  
     
     
         42 . The method according to  claim 41  wherein said detecting is carried out by naked eye observation of a user.  
     
     
         43 . The method according to  claim 33  wherein the first and second single-stranded oligonucleotide probes comprise the same nucleotide sequence.  
     
     
         44 . The method according to  claim 33  wherein the first and second single-stranded oligonucleotide probes comprise different nucleotide sequences.  
     
     
         45 . The method according to  claim 33  wherein the plurality of metal nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles, and combinations thereof.  
     
     
         46 . The method according to  claim 33  wherein the target nucleic acid is DNA.  
     
     
         47 . The method according to  claim 33  wherein the target nucleic acid is RNA.  
     
     
         48  The method according to  claim 33  wherein the target nucleic acid is obtained from a human, a virus, a bacteria, an animal, an insect, or a plant.  
     
     
         49 . The method according to  claim 33  wherein the target nucleic acid is a single-stranded product of a polymerase chain reaction.  
     
     
         50 . The method according to  claim 33  wherein the test solution is obtained from a polymerase chain reaction and is exposed to denaturing conditions prior to said combining.  
     
     
         51 . The method according to  claim 33  wherein each of the first and second single-stranded oligonucleotide probes is between about 10 and about 50 nucleotides in length.  
     
     
         52 . The method according to  claim 33  wherein the at least one first single-stranded oligonucleotide probe and the at least one second single-stranded oligonucleotide probe are substantially the same length.  
     
     
         53 . The method according to  claim 33  wherein one or both of the first and second single-stranded oligonucleotide probes comprises structurally modified DNA or RNA.  
     
     
         54 . A method for detecting a single nucleotide polymorphism in a target nucleic acid molecule, said method comprising: 
 combining (i) a solution comprising a target nucleic acid molecule and (ii) at least one first single-stranded oligonucleotide probe comprising a nucleotide sequence and a fluorescent label attached thereto, wherein the nucleotide sequence hybridizes to a region of the target nucleic acid molecule that may contain a single-nucleotide polymorphism, to form a hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one first single-stranded oligonucleotide probe to form at least one hybridization complex;    exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow unhybridized probes in the hybridization solution to electrostatically associate with the metal nanoparticles;    determining a temperature of the hybridization solution where quenching of the photoluminescence by the fluorescent label begins, said temperature representing the melting temperature; and    comparing the melting temperature for the hybridization solution with a known melting temperature of a perfectly complementary probe,    wherein a difference between the melting temperatures indicates the presence of the single nucleotide polymorphism in the target nucleic acid molecule.    
     
     
         55 . The method according to  claim 54  wherein the fluorescent label is selected from the group consisting of an organic dye, semiconductor quantum dots, lanthanide atom-containing complexes, and a fluorescent protein.  
     
     
         56 . The method according to  claim 54  wherein the plurality of metal nanoparticles are introduced into the hybridization solution at a concentration of between about 1 and about 100 nM.  
     
     
         57 . The method according to  claim 54  wherein at least one single-stranded oligonucleotide probe is present in the hybridization solution at a concentration of about 1 to about 100,000 nM/ml.  
     
     
         58 . The method according to  claim 54  wherein the plurality of metal nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles, and combinations thereof.  
     
     
         59 . The method according to  claim 54  wherein the target nucleic acid is DNA.  
     
     
         60 . The method according to  claim 54  wherein the target nucleic acid is RNA.  
     
     
         61  The method according to  claim 54  wherein the target nucleic acid is obtained from a human, a virus, a bacteria, an animal, an insect, or a plant.  
     
     
         62 . The method according to  claim 54  wherein the target nucleic acid is a single-stranded product of a polymerase chain reaction.  
     
     
         63 . The method according to  claim 54  wherein the test solution is obtained from a polymerase chain reaction and is exposed to denaturing conditions prior to said combining.  
     
     
         64 . The method according to  claim 54  wherein the single-stranded oligonucleotide probe is between about 10 and about 50 nucleotides in length.  
     
     
         65 . The method according to  claim 54  wherein the single-stranded oligonucleotide probes comprises structurally modified DNA or RNA.  
     
     
         66 . A kit comprising: 
 a first container that contains a colloidal solution comprising metal nanoparticles;    a second container that contains an aqueous solution comprising at least one single-stranded oligonucleotide probe comprising a nucleotide sequence that is substantially complementary to a target nucleic acid molecule.    
     
     
         67 . The kit according to  claim 66  wherein the metal nanoparticles are characterized by a surface that is substantially free of conjugated nucleic acid molecules.  
     
     
         68 . The kit according to  claim 66  wherein the metal nanoparticles are between about 5 and about 500 nm in diameter.  
     
     
         69 . The kit according to  claim 66  wherein the metal nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles, and combinations thereof.  
     
     
         70 . The kit according to  claim 66  further comprising a containing that contains a salt solution comprises a Na+concentration of between about 0.01 and about 5 M.  
     
     
         71 . The kit according to  claim 66  wherein at least one single-stranded oligonucleotide probe is present in the aqueous solution at a concentration of about 1 to about 100,000 nM.  
     
     
         72 . The kit according to  claim 66  wherein the at least one single-stranded oligonucleotide probe is conjugated to a fluorescent label.  
     
     
         73 . The kit according to  claim 72  wherein the fluorescent label is selected from the group consisting of an organic dye, semiconductor quantum dots, lanthanide atom-containing complexes, and a fluorescent protein.  
     
     
         74 . The kit according to  claim 66  wherein the third container comprises at least two single-stranded oligonucleotide probes.  
     
     
         75 . The kit according to  claim 74  wherein each of the at least two single-stranded oligonucleotide probes is conjugated to a fluorescent label, the fluorescent label being different for the at least two single-stranded oligonucleotide probes.  
     
     
         76 . The kit according to  claim 66  wherein each of the at least one single-stranded oligonucleotide probes is between about 10 and about 50 nucleotides in length.  
     
     
         77 . The kit according to  claim 66  wherein at least one single-stranded oligonucleotide probe comprises structurally modified DNA or RNA.  
     
     
         78 . The kit according to  claim 66  wherein the at least one single-stranded oligonucleotide probe comprises a nucleotide sequence that is 100 percent complementary to the target nucleic acid.  
     
     
         79 . A method for detecting a target nucleic acid in a test solution comprising: 
 subjecting a portion of a test solution potentially including a target nucleic acid to polymerase chain reaction and obtaining a product solution comprising single-stranded nucleic acid products of the polymerase chain reaction;    combining at least one single-stranded oligonucleotide probe with the product solution to form a hybridization solution under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the product solution;    exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow any single-stranded nucleic acids in the hybridization solution to associate with the plurality of metal nanoparticles; and    determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with one or more of the plurality of metal nanoparticles, wherein hybridization to the target nucleic acid or electrostatic association with one or more metal nanoparticles is indicated by an optical property of the hybridization solution.    
     
     
         80 . The method according to  claim 79  wherein the plurality of metal nanoparticles are introduced into the hybridization solution at a concentration of between about 1 and about 100 nM.  
     
     
         81 . The method according to  claim 79  wherein the plurality of metal nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, platinum nanoparticles, and combinations thereof.  
     
     
         82 . The method according to  claim 79  wherein at least one single-stranded oligonucleotide probe is present in the hybridization solution at a concentration of about 1 to about 100,000 nM.  
     
     
         83 . The method according to  claim 79  wherein said determining comprises: 
 detecting a color change of the solution after said exposing, whereby a color change indicates substantial aggregation of the plurality of metal nanoparticles in the absence of a target nucleic acid.    
     
     
         84 . The method according to  claim 79  wherein the at least one single-stranded oligonucleotide probe is conjugated to a fluorescent label.  
     
     
         85 . The method according to  claim 84  wherein the fluorescent label is selected from the group consisting of an organic dye, semiconductor quantum dots, lanthanide atom-containing complex, and a fluorescent protein.  
     
     
         86 . The method according to  claim 79  wherein said determining comprises: 
 detecting photoluminescence caused by the fluorescent label after said exposing, which presence of a target nucleic acid.    
     
     
         87 . The method according to  claim 79  wherein hybridization complexes in the hybridization solution do not significantly associate electrostatically with the plurality of metal nanoparticles.  
     
     
         88 . The method according to  claim 79  wherein the target nucleic acid is cDNA and the polymerase chain reaction is a reverse transcription-polymerase chain reaction.  
     
     
         89 . The method according to  claim 79  wherein target nucleic acid is synthetic, natural, or structurally modified DNA or RNA.  
     
     
         90 . The method according to  claim 79  wherein the target nucleic acid comprises a nucleic acid molecule coupled to a protein or polypeptide.  
     
     
         91 . The method according to  claim 79  wherein the polymerase chain reaction is an immuno-polymerase chain reaction.  
     
     
         92 . A method of detecting a pathogen in a sample comprising: 
 obtaining a sample that may contain nucleic acid of a pathogen; and    performing the method of  claim 1 , wherein said determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates presence of the pathogen.    
     
     
         93 . The method according to  claim 92  wherein the nucleic acid isolated from the sample is RNA and the target nucleic acid is RNA.  
     
     
         94 . The method according to  claim 92  wherein the nucleic acid isolated from the sample is RNA and the target nucleic acid is cDNA, said method further comprising: 
 amplifying the isolated RNA by reverse-transcription polymerase chain reaction prior to said performing.    
     
     
         95 . A method of genetic screening comprising: 
 obtaining a sample;    isolating DNA from the sample;    amplifying the DNA isolated from the sample; and    performing the method of  claim 1 , wherein said determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates predisposition to a genetic condition, hereditary condition, or identifies an organism.    
     
     
         96 . A method of detecting a protein in a sample comprising: 
 obtaining a sample;    performing an immuno-polymerase chain reaction procedure using the sample, wherein the immuno-polymerase chain reaction procedure results in amplification of a nucleic acid conjugated to a protein; and    performing the method of  claim 1 , wherein the nucleic acid that is conjugated to the protein is the target nucleic acid, and wherein said determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates that the protein is present in the sample.    
     
     
         97 . A method of quantifying the amount of amplified nucleic acid prepared by polymerase chain reaction, said method comprising: 
 providing two or more fluorescently labeled oligonucleotide primers that each comprise a nucleotide sequence capable of hybridizing to a nucleic acid molecule, or its complement, to be amplified;    performing polymerase chain reaction using a target nucleic acid molecule and/or its complement, and the provided fluorescently labeled oligonucleotide primers; and    performing the method of  claim 1  on a sample obtained after said performing polymerase chain reaction, wherein the level of fluorescence detected from the sample indicates the amount of primer that has been incorporated into an amplified nucleic acid molecule.